ABSTRACT Colorectal cancer is the 4th most incident cancer in the U.S. and the second leading cause of cancer mortality, representing an unmet clinical need for prevention strategies. In this context, a novel tumor suppressor, guanylate cyclase C (GUCY2C), offers an opportunity to advance the prevention and treatment of colorectal cancer. GUCY2C is a transmembrane receptor expressed on the luminal aspect of the intestinal epithelium. Together with its endogenous ligand, guanylin, it regulates intestinal fluid secretion, tissue architecture, and several canonical cancer signaling networks. Intriguingly, guanylin is among the most commonly lost gene products in sporadic colorectal cancer. Its loss occurs early in transformation and arises in lesions harboring mutations of adenomatous polyposis coli (APC) or its downstream target, ?-catenin, which are responsible for >90% of sporadic colorectal cancers. These mutations lead to stabilization and nuclear accumulation of ?- catenin, driving a transcriptional program underlying tumorigenesis. While aberrant APC/?-catenin signaling is a well-established driver of colorectal cancer, downstream mechanisms regulating transformation remain incompletely understood. Our preliminary data indicate that enforced transgenic guanylin expression eliminates the development of tumors in mouse models of APC/?-catenin-driven cancer, suggesting a link between these two signaling axes. Hence, we hypothesize that colorectal cancer driven by aberrant APC/?-catenin signaling reflects guanylin loss, which silences the GUCY2C tumor-suppressor. This project will explore the role of guanylin loss in colorectal cancer, with the ultimate intent to demonstrate the utility of GUCY2C ligand replacement as a means of colorectal cancer prevention. To this end, we propose two specific aims. In Aim 1, we will define guanylin loss as a required step in APC/?-catenin-dependent intestinal tumorigenesis, which can be overcome by hormone replacement therapy. Using conditional mouse models of intestinal cancer developed by our laboratory, we will induce biallelic APC loss or ?-catenin activating mutations and examine the potential for transgenic guanylin or oral GUCY2C ligands to oppose tumorigenesis. Results will inform chemopreventive strategies to prevent colorectal cancer. In Aim 2, we will elucidate the mechanisms by which APC/?-catenin signaling suppresses guanylin. Using human cancer cells and mouse models of colorectal cancer, we will define the transcriptional mechanisms by which APC/?-catenin suppress guanylin, permitting tumorigenesis. Results will reveal reversible mechanisms contributing to colorectal cancer (guanylin loss) that would expand the pathophysiological model of intestinal tumorigenesis. These studies will have immediate translational potential for patients harboring APC/?-catenin mutations (>90% of colorectal cancers), due to the recent FDA-approval of the oral GUCY2C ligands, linaclotide (LinzessTM) or plecanatide (TrulanceTM), which could be leveraged for hormone replacement therapy.